Glucocorticoids attenuate interleukin-6-induced c-Fos and Egr1 expression and impair neuritogenesis in PC12 cells.

Interleukin-6 (IL-6) is a cytokine primarily known for immune regulation. There is also growing evidence that IL-6 triggers neurogenesis and impacts neural development, both life-long occurring processes that can be impaired by early-life and adult stress. Stress induces the release of glucocorticoids by activation of the hypothalamic-pituitary-adrenal (HPA) axis. On the cellular level, glucocorticoids act via the ubiquitously expressed glucocorticoid receptor. Thus, we aimed to elucidate whether glucocorticoids affect IL-6-induced neural development. Here, we show that IL-6 signalling induces neurite outgrowth in adrenal pheochromocytoma PC12 cells in a mitogen-activated protein kinase (MAPK) pathway-dependent manner, since neurite outgrowth was diminished upon Mek-inhibitor treatment. Using quantitative biochemical approaches, such as qRT-PCR analysis of Hyper-IL-6 treated PC12 cells, we show that neurite outgrowth induced by IL-6 signalling is accompanied by early and transient MAPK-dependent mRNA expression of immediate early genes coding for proteins such as early growth response protein 1 (Egr1) and c-Fos. This correlates with reduced proliferation and prolonged G0/G1 cell cycle arrest as determined by monitoring the cellular DNA content using flow cytometry. These results indicate for IL-6 signalling-induced neural differentiation. Interestingly, the glucocorticoid Dexamethasone impairs early IL-6 signalling-induced mRNA expression of c-Fos and Egr1 and restrains neurite outgrowth. Impaired Egr1 and c-Fos expression in neural development is implicated in the aetiology of neuropathologies. Thus, it appears likely that stress-induced release of glucocorticoids, as well as therapeutically administered glucocorticoids, contribute to the development of neuropathologies by reducing the expression of Egr1 and c-Fos, and by restraining IL-6-dependent neural differentiation.

The tyrosine phosphatase SHP2 increases robustness and information transfer within IL-6-induced JAK/STAT signalling.

BACKGROUND: Cell-to-cell heterogeneity is an inherent feature of multicellular organisms and is central in all physiological and pathophysiological processes including cellular signal transduction. The cytokine IL-6 is an essential mediator of pro- and anti-inflammatory processes. Dysregulated IL-6-induced intracellular JAK/STAT signalling is associated with severe inflammatory and proliferative diseases. Under physiological conditions JAK/STAT signalling is rigorously controlled and timely orchestrated by regulatory mechanisms such as expression of the feedback-inhibitor SOCS3 and activation of the protein-tyrosine phosphatase SHP2 (PTPN11). Interestingly, the function of negative regulators seems not to be restricted to controlling the strength and timely orchestration of IL-6-induced STAT3 activation. Exemplarily, SOCS3 increases robustness of late IL-6-induced STAT3 activation against heterogenous STAT3 expression and reduces the amount of information transferred through JAK/STAT signalling. METHODS: Here we use multiplexed single-cell analyses and information theoretic approaches to clarify whether also SHP2 contributes to robustness of STAT3 activation and whether SHP2 affects the amount of information transferred through IL-6-induced JAK/STAT signalling. RESULTS: SHP2 increases robustness of both basal, cytokine-independent STAT3 activation and early IL-6-induced STAT3 activation against differential STAT3 expression. However, SHP2 does not affect robustness of late IL-6-induced STAT3 activation. In contrast to SOCS3, SHP2 increases the amount of information transferred through IL-6-induced JAK/STAT signalling, probably by reducing cytokine-independent STAT3 activation and thereby increasing sensitivity of the cells. These effects are independent of SHP2-dependent MAPK activation. CONCLUSION: In summary, the results of this study extend our knowledge of the functions of SHP2 in IL-6-induced JAK/STAT signalling. SHP2 is not only a repressor of basal and cytokine-induced STAT3 activity, but also ensures robustness and transmission of information. Plain English summary Cells within a multicellular organism communicate with each other to exchange information about the environment. Communication between cells is facilitated by soluble molecules that transmit information from one cell to the other. Cytokines such as interleukin-6 are important soluble mediators that are secreted when an organism is faced with infections or inflammation. Secreted cytokines bind to receptors within the membrane of their target cells. This binding induces activation of an intracellular cascade of reactions called signal transduction, which leads to cellular responses. An important example of intracellular signal transduction is JAK/STAT signalling. In healthy organisms signalling is controlled and timed by regulatory mechanisms, whose activation results in a controlled shutdown of signalling pathways. Interestingly, not all cells within an organism are identical. They differ in the amount of proteins involved in signal transduction, such as STAT3. These differences shape cellular communication and responses to intracellular signalling. Here, we show that an important negative regulatory protein called SHP2 (or PTPN11) is not only responsible for shutting down signalling, but also for steering signalling in heterogeneous cell populations. SHP2 increases robustness of STAT3 activation against variable STAT3 amounts in individual cells. Additionally, it increases the amount of information transferred through JAK/STAT signalling by increasing the dynamic range of pathway activation in heterogeneous cell populations. This is an amazing new function of negative regulatory proteins that contributes to communication in heterogeneous multicellular organisms in health and disease. Video Abstract.

Intragenic regulation of SOCS3 isoforms.

BACKGROUND: Inflammatory reactions are commonly affected by stress responses. Interleukin-6 signalling is part of the inflammatory response and is stringently regulated by the feedback inhibitor SOCS3 expressed in a short and long isoform. Here, we studied the inhibitory potential of the two SOCS3 isoforms. Furthermore, we analysed the regulation of SOCS3 isoform expression and the role of PKR stress kinase signalling in SOCS3 protein expression. METHODS: We performed Western blotting, reporter assays, genetic analyses and manipulations for studying SOCS3 isoform expression and activation of signalling components involved in interleukin-6-induced and PKR-dependent signalling. RESULTS: Interleukin-6-induced endogenous expression of both SOCS3 isoforms was found in distinct cell types. Forced expression of either the long or short SOCS3 isoform demonstrated equal inhibitory activity of each isoform and confirmed longer half-life of the short isoform. Study of intragenic regulation of SOCS3 isoform expression revealed that (i) the 5'-UTR of SOCS3 mRNA restrains specifically expression of the long SOCS3 isoform, (ii) expression of the long isoform restrains expression of the short isoform, and (iii) signalling through the stress kinase PKR does not impact on SOCS3 isoform ratio. CONCLUSIONS: Both SOCS3 isoforms show a similar potential for inhibiting interleukin-6 signalling but differ in their half-lives. Relative expression of the isoforms depends on intragenic elements yet is independent of PKR signalling.

The multi-site docking protein Grb2-associated binder 1 (Gab1) enhances interleukin-6-induced MAPK-pathway activation in an SHP2-, Grb2-, and time-dependent manner.

BACKGROUND: Cytokine-dependent activation of signalling pathways is tightly orchestrated. The spatiotemporal activation of signalling pathways dictates the specific physiological responses to cytokines. Dysregulated signalling accounts for neoplastic, developmental, and inflammatory diseases. Grb2-associated binder (Gab) family proteins are multi-site docking proteins, which expand cytokine-induced signal transduction in a spatial- and time-dependent manner by coordinating the recruitment of proteins involved in mitogen activated protein kinase (MAPK)/extracellular-signal regulated kinase (ERK) and phosphatidyl-inositol-3-kinase (PI3K) signalling. Interaction of Gab family proteins with these signalling proteins determines strength, duration and localization of active signalling cascades. However, the underlying molecular mechanisms of signal orchestration by Gab family proteins in IL-6-induced signalling are only scarcely understood. METHODS: We performed kinetic analyses of interleukin-6 (IL-6)-induced MAPK activation and analysed downstream responses. We compared signalling in wild-type cells, Gab1 knock-out cells, those reconstituted to express Gab1 mutants, and cells expressing gp130 receptors or receptor mutants. RESULTS: Interleukin-6-induced MAPK pathway activation can be sub-divided into an early Gab1-independent and a subsequent Gab1-dependent phase. Early Gab1-independent MAPK activation is critical for the subsequent initiation of Gab1-dependent amplification of MAPK pathway activation and requires binding of SH2 domain-containing phosphatase 2 (SHP2) to the interleukin-6 receptor complex. Subsequent and coordinated recruitment of Grb2 and SHP2 to Gab1 is essential for Gab1-dependent amplification of IL-6-induced late MAPK pathway activation and subsequent gene expression. CONCLUSIONS: Overall, we elaborated the molecular requirements for Gab1-dependent, spatiotemporal orchestration of interleukin-6-dependent MAPK signalling. We discriminated IL-6-induced Gab1-independent, early activation of MAPK signalling and Gab1-dependent, sustained activation of MAPK signalling.

Response to IL-6 trans- and IL-6 classic signalling is determined by the ratio of the IL-6 receptor α to gp130 expression: fusing experimental insights and dynamic modelling.

BACKGROUND: Interleukin-6 is a pleiotropic cytokine with high clinical relevance and an important mediator of cellular communication, orchestrating both pro- and anti-inflammatory processes. Interleukin-6-induced signalling is initiated by binding of IL-6 to the IL-6 receptor α and subsequent binding to the signal transducing receptor subunit gp130. This active receptor complex initiates signalling through the Janus kinase/signal transducer and activator of transcription pathway. Of note, IL-6 receptor α exists in a soluble and a transmembrane form. Binding of IL-6 to membrane-bound IL-6 receptor α induces anti-inflammatory classic signalling, whereas binding of IL-6 to soluble IL-6 receptor α induces pro-inflammatory trans-signalling. Trans-signalling has been described to be markedly stronger than classic signalling. Understanding the molecular mechanisms that drive differences between trans- and classic signalling is important for the design of trans-signalling-specific therapies. These differences will be addressed here using a combination of dynamic mathematical modelling and molecular biology. METHODS: We apply an iterative systems biology approach using set-based modelling and validation approaches combined with quantitative biochemical and cell biological analyses. RESULTS: The combination of experimental analyses and dynamic modelling allows to relate the observed differences between IL-6-induced trans- and classic signalling to cell-type specific differences in the expression and ratios of the individual subunits of the IL-6 receptor complex. Canonical intracellular Jak/STAT signalling is indifferent in IL-6-induced trans- and classic signalling. CONCLUSION: This study contributes to the understanding of molecular mechanisms of IL-6 signal transduction and underlines the power of combined dynamical modelling, model-based validation and biological experiments. The opposing pro- and anti-inflammatory responses initiated by IL-6 trans- and classic signalling depend solely on the expression ratios of the subunits of the entire receptor complex. By pointing out the importance of the receptor expression ratio for the strength of IL-6 signalling this study lays a foundation for future precision medicine approaches that aim to selectively block pro-inflammatory trans-signalling. Furthermore, the derived models can be used for future therapy design.

Self-organization and regulation of intrinsically disordered proteins with folded N-termini.

How do mostly disordered proteins coordinate the specific assembly of very large signal transduction protein complexes? A newly emerging hypothesis may provide some clues towards a molecular mechanism.

Glucocorticoids increase interleukin-6-dependent gene induction by interfering with the expression of the suppressor of cytokine signaling 3 feedback inhibitor.

UNLABELLED: Glucocorticoids are known to be potent regulators of inflammation and have been used pharmacologically against inflammatory, immune, and lymphoproliferative diseases for more than 50 years. Due to their possible and well-documented side effects, it is crucial to understand the molecular mechanisms and targets of glucocorticoid action in detail. Several modes of action have been discussed; nevertheless, none of them fully explain all the functions of glucocorticoids. Therefore, we analyzed the cross-talk between glucocorticoids and interleukin-6 (IL-6) in the liver. IL-6 exerts pro-inflammatory as well as anti-inflammatory properties and is a main inducer of the acute-phase response. The balance between the proinflammatory and anti-inflammatory activities of IL-6 is tightly regulated by suppressor of cytokine signaling 3 (SOCS3), a well-known feedback inhibitor of IL-6 signaling. Here, it is demonstrated that glucocorticoids enhance IL-6-dependent γ-fibrinogen expression. Studying of the underlying mechanism revealed prolonged activation of signal transducer and activator of transcription 3 (STAT3) caused by down-regulation of SOCS3 protein expression. Consequently, in SOCS3-deficient cells glucocorticoids do not affect IL-6-induced signal transduction. Moreover, in hepatocytes lacking the SOCS3 recruiting motif within gp130, IL-6-dependent γ-fibrinogen expression is not influenced by glucocorticoid treatment. CONCLUSION: Glucocorticoids interfere with IL-6-induced expression of the feedback inhibitor SOCS3, thereby leading to enhanced expression of acute-phase genes in hepatocytes. This mechanism contributes to the explanation of how glucocorticoids affect inflammation and acute-phase gene induction.

Non-canonical STAT3 function reduces REDD1 transcription.

The transcription factor STAT3 is a potent activator of transcription, but evidence exists that STAT3 can also repress gene expression. However, little is known about the molecular mechanisms involved in STAT3-dependent gene repression. Notably, STAT3 reduces the expression of the stress-induced mTOR inhibitor REDD1 by reducing REDD1 mRNA transcription. Here, we determined the functional domains of STAT3 responsible for the reduction of REDD1 mRNA and protein expression. Within STAT3, the N-terminal domain and tyrosine 705 are crucial for STAT3-dependent reduction of REDD1 expression. Interestingly, binding of STAT3 to canonical STAT-binding sides within the REDD1 promoter is not necessary for STAT3-mediated reduction of REDD1 expression. Still, STAT3 is recruited to the REDD1 promoter upon stimulation with IL-6, and reduces REDD1 promoter activity. The reduction of REDD1 expression is specific for STAT3, as neither expression nor activation of STAT1 reduces REDD1 mRNA and protein expression. In summary, we present a novel, non-canonical STAT3-dependent mechanism for reducing gene expression. This transcriptional repression increases the functions of STAT3 proteins beyond classical transcriptional activation of cytokine-regulated target genes to a more complex function in modulating gene expression in immunity and cellular stress.

Cross-regulation of cytokine signalling: pro-inflammatory cytokines restrict IL-6 signalling through receptor internalisation and degradation.

The inflammatory response involves a complex interplay of different cytokines which act in an auto- or paracrine manner to induce the so-called acute phase response. Cytokines are known to crosstalk on multiple levels, for instance by regulating the mRNA stability of targeted cytokines through activation of the p38-MAPK pathway. In our study we discovered a new mechanism that answers the long-standing question how pro-inflammatory cytokines and environmental stress restrict immediate signalling of interleukin (IL)-6-type cytokines. We show that p38, activated by IL-1beta, TNFalpha or environmental stress, impairs IL-6-induced JAK/STAT signalling through phosphorylation of the common cytokine receptor subunit gp130 and its subsequent internalisation and degradation. We identify MK2 as the kinase that phosphorylates serine 782 in the cytoplasmic part of gp130. Consequently, inhibition of p38 or MK2, deletion of MK2 or mutation of crucial amino acids within the MK2 target site or the di-leucine internalisation motif blocks receptor depletion and restores IL-6-dependent STAT activation as well as gene induction. Hence, a novel negative crosstalk mechanism for cytokine signalling is described, where cytokine receptor turnover is regulated in trans by pro-inflammatory cytokines and stress stimuli to coordinate the inflammatory response.

Glucagon counteracts interleukin-6-dependent gene expression by redundant action of Epac and PKA.

Inflammation is the biological response to injurious stimuli. In the initial phase of the inflammatory process, interleukin-6 (IL-6) is the main inducer of acute phase protein expression in the liver. A prolonged acute phase response is characterised by a disturbed glucose homeostasis and elevated levels of IL-6, insulin, and counterregulatory hormones such as glucagon. Several studies deal with the impact of IL-6 on glucagon-dependent gene expression. In contrast, only very little is known about the influence of G-protein-coupled receptors on IL-6 signalling. Therefore, the aim of this study is to elucidate the regulation of IL-6-induced gene expression by glucagon. We could reveal a novel mechanism of negative regulation of IL-6-induced MAP kinase activation by glucagon in primary murine hepatocytes. IL-6-dependent induction of the ERK-dependent target gene Tfpi2, coding for a Kunitz-type serine protease inhibitor, was strongly down-regulated by glucagon treatment. Studying the underlying mechanism revealed a redundant action of the signalling molecules exchange protein activated by cyclic AMP (Epac) and protein kinase A. The metabolic hormone glucagon interferes in IL-6-induced gene expression. This observation is indicative for a regulatory role of G-protein-coupled receptors in the IL-6-dependent inflammatory response.

Signal transduction in the footsteps of goethe and schiller.

The historical town of Weimar in Thuringia, the "green heart of Germany" was the sphere of Goethe and Schiller, the two most famous representatives of German literature's classic era. Not yet entirely as influential as those two cultural icons, the Signal Transduction Society (STS) has nevertheless in the last decade established within the walls of Weimar an annual interdisciplinary Meeting on "Signal Transduction - Receptors, Mediators and Genes", which is well recognized as a most attractive opportunity to exchange results and ideas in the field.The 12th STS Meeting was held from October 28 to 31 and provided a state-of-the-art overview of various areas of signal transduction research in which progress is fast and discussion lively. This report is intended to share with the readers of CCS some highlights of the Meeting Workshops devoted to specific aspects of signal transduction.

SHPS-1/SIRP1alpha contributes to interleukin-6 signalling.

The transmembrane glycoprotein signal regulatory protein/SHP2-substrate (SIRP1alpha/SHPS-1) has been implicated in growth factor- and cell adhesion-induced signalling. Here we report on the contribution of SIRP1alpha to IL-6 type cytokine signalling. SIRP1alpha binds the protein tyrosine phosphatase SHP2 upon treatment with interleukin-6 in a stimulation-dependent manner. Mouse embryonic fibroblasts expressing a SIRP1alpha protein which lacks the intracellular part show enhanced SHP2 phosphorylation and ERK1/2 activation in response to IL-6, suggesting that SIRP1alpha affects IL-6-signalling through SHP2. Whereas SHP2 phosphorylation is enhanced in SIRP1alpha-deficient cells STAT3 activation is delayed and STAT3-dependent gene induction is reduced which correlates with reduced STAT3 serine phosphorylation. Our results indicate that SIRP1alpha contributes to IL-6 signalling by counteracting SHP2 phosphorylation which consequently affects ERK-activation and STAT3-dependent transactivation as well as target gene expression. Our observations will help to understand the tight balance of MAPK- and STAT3-activation in response to IL-6 which was found to be misbalanced in many autoimmune diseases, inflammatory proliferative diseases and cancer.

Interleukin-6 acts in the fashion of a classical chemokine on monocytic cells by inducing integrin activation, cell adhesion, actin polymerization, chemotaxis, and transmigration.

Macrophages contribute to the innate immune response by eliminating bacteria, viral particles, and apoptotic bodies. They develop from circulating monocytes. In case of an infection, monocytes attach to the endothelial cells of the blood vessels, migrate along the endothelial cells, leave the circulatory system to enter the inflammatory tissue, and differentiate into macrophages. Cell migration is induced frequently by chemokines that act through G-protein-coupled receptors. Only a few cytokines signaling through single-transmembrane domain receptors have been shown to induce cell migration. Often, this potential depends on the induction of classical chemokines and is not a direct cellular effect. Here, we discovered IL-6 as a potent stimulant for monocytic cell migration. Furthermore, we present data about IL-6-induced integrin activation, cell attachment, actin polymerization, fibronectin-dependent migration, and transmigration through a layer of endothelial cells. Our results show that IL-6 fulfills all biological properties to mediate cell migration of monocytic cells, which may contribute to the proinflammatory potential of IL-6.

Prostaglandin E1 inhibits IL-6-induced MCP-1 expression by interfering specifically in IL-6-dependent ERK1/2, but not STAT3, activation.

IL (interleukin)-6 exerts pro- as well as anti-inflammatory activities. Beside many other activities, IL-6 is the major inducer of acute phase proteins in the liver, acts as a differentiation factor for blood cells, as migration factor for T-cells and is a potent inducer of the chemokine MCP-1 (monocyte chemoattractant protein-1). Recent studies have focused on the negative regulation of IL-6 signal transduction through the IL-6-induced feedback inhibitors SOCS (suppressor of cytokine signalling) 1 and SOCS3 or the protein tyrosine phosphatases SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) and TcPTP (T-cell protein tyrosine phosphatase). Studies on the cross-talk between pro-inflammatory mediators (IL-1, tumour necrosis factor, lipopolysaccharide) and IL-6 elucidated further regulatory mechanisms. Less is known about the regulation of IL-6 signal transduction by hormone/cytokine signalling through G-protein-coupled receptors. This is particularly surprising since many of these hormones (such as prostaglandins and chemokines) play an important role in inflammatory processes. In the present study, we have investigated the inhibitory activity of PGE(1) (prostaglandin E(1)) on IL-6-induced MCP-1 expression and have elucidated the underlying molecular mechanism. Surprisingly, PGE(1) does not affect IL-6-induced STAT (signal transducer and activator of transcription) 3 activation, but does affect ERK (extracellular-signal-regulated kinase) 1/2 activation which is crucial for IL-6-dependent expression of MCP-1. In summary, we have discovered a specific cross-talk between the adenylate cyclase cascade and the IL-6-induced MAPK (mitogen-activated protein kinase) cascade and have investigated its impact on IL-6-dependent gene expression.

Robustness and Information Transfer within IL-6-induced JAK/STAT Signalling.

Cellular communication via intracellular signalling pathways is crucial. Expression and activation of signalling proteins is heterogenous between isogenic cells of the same cell-type. However, mechanisms evolved to enable sufficient communication and to ensure cellular functions. We use information theory to clarify mechanisms facilitating IL-6-induced JAK/STAT signalling despite cell-to-cell variability. We show that different mechanisms enabling robustness against variability complement each other. Early STAT3 activation is robust as long as cytokine concentrations are low. Robustness at high cytokine concentrations is ensured by high STAT3 expression or serine phosphorylation. Later the feedback-inhibitor SOCS3 increases robustness. Channel Capacity of JAK/STAT signalling is limited by cell-to-cell variability in STAT3 expression and is affected by the same mechanisms governing robustness. Increasing STAT3 amount increases Channel Capacity and robustness, whereas increasing STAT3 tyrosine phosphorylation reduces robustness but increases Channel Capacity. In summary, we elucidate mechanisms preventing dysregulated signalling by enabling reliable JAK/STAT signalling despite cell-to-cell heterogeneity.

Activation of NF-kappaB by IL-1beta blocks IL-6-induced sustained STAT3 activation and STAT3-dependent gene expression of the human gamma-fibrinogen gene.

Despite the essential role of the fibrinogen gamma-chain as a blood clotting factor, the fibrinogen gamma-chain contains a number of interaction sites to recruit other factors such as leukocytes important for prevention of pathogen entry and propagation of the repair process. Interleukin-6 (IL-6) is known as the major inducer of gamma-fibrinogen synthesis in hepatocytes, whereas IL-1beta has been shown to act as a potent inhibitor of gamma-fibrinogen expression. Studies on the rat fibrinogen gamma-chain promoter suggest that nuclear factor (NF)-kappaB replaces the signal transducer and activator of transcription (STAT) 3 from binding to overlapping NF-kappaB/STAT3 binding sites within the 5' regulatory region of the rat gamma-chain gene promoter. However, despite its physiological relevance, the underlying mechanism responsible for the inhibitory effect of IL-1beta in humans is still not understood and apparently more complex. In contrast to the mechanism described for the rat gene our results indicate that IL-1beta suppresses the IL-6-induced activation of the human gamma-fibrinogen gene particularly by blocking the late phase STAT3-tyrosine phosphorylation NF-kappaB-dependently but independent from de novo protein synthesis. Consequently, blocking NF-kappaB activation restores specifically late phase STAT3 activation as well as the induction of the human gamma-fibrinogen gene. In contrast, specifically early STAT3 activation could be restored by a block of the p38 mitogen-activated protein kinase (p38(MAPK)) pathway. In summary, our results indicate that expression of the gamma-fibrinogen gene is mainly controlled by the strength of late phase STAT3 activation, which in turn is negatively regulated by the extent of IL-1beta-mediated NF-kappaB activity.

Designing optimal experiments to discriminate interaction graph models.

Modern methods for the inference of cellular networks from experimental data often express nondeterminism through an ensemble of candidate models. To discriminate among these candidates new experiments need to be carried out. Theoretically, the number of possible experiments is exponential in the number of possible perturbations. In praxis, experiments are expensive and there exist several limiting constraints. Limiting factors exist on the combinations of perturbations that are technically possible, which components can be measured, and on the number of affordable experiments. Further, not all experiments are equally well suited to discriminate model candidates. The goal of optimal experiment design is to determine those experiments that discriminate most of the candidates while minimizing the costs. We present an approach for experiment planning with interaction graph models and sign consistency methods. This new approach can be used in combination with methods for network inference and consistency checking. We applied our method to study the Erythropoietin signal transduction in human kidney cells HEK293. We first used simulated experiment data from an ODE model to demonstrate in silico that our experimental design results in the inference of the gold standard model. Finally, we used the approach to plan in vivo experiments that discriminate model candidates for the Erythropoietin signal transduction in this cell line.

Interaction of suppressor of cytokine signalling 3 with cavin-1 links SOCS3 function and cavin-1 stability.

Effective suppression of JAK-STAT signalling by the inducible inhibitor "suppressor of cytokine signalling 3" (SOCS3) is essential for limiting signalling from cytokine receptors. Here we show that cavin-1, a component of caveolae, is a functionally significant SOCS3-interacting protein. Biochemical and confocal imaging demonstrate that SOCS3 localisation to the plasma membrane requires cavin-1. SOCS3 is also critical for cavin-1 stabilisation, such that deletion of SOCS3 reduces the expression of cavin-1 and caveolin-1 proteins, thereby reducing caveola abundance in endothelial cells. Moreover, the interaction of cavin-1 and SOCS3 is essential for SOCS3 function, as loss of cavin-1 enhances cytokine-stimulated STAT3 phosphorylation and abolishes SOCS3-dependent inhibition of IL-6 signalling by cyclic AMP. Together, these findings reveal a new functionally important mechanism linking SOCS3-mediated inhibition of cytokine signalling to localisation at the plasma membrane via interaction with and stabilisation of cavin-1.

Determinants governing the potency of STAT3 activation via the individual STAT3-recruiting motifs of gp130.

In recent years, the elucidation of the structures of many signalling molecules has allowed new insights into the molecular mechanisms that govern signal transduction events. In the field of cytokine signalling, the solved structures of cytokine/receptor complexes and of key components involved in signal transduction such as STAT factors or the tyrosine phosphatase SHP2 have broadened our understanding of the molecular basis of the signalling events and provided key information for the rational design of therapeutic approaches to modulate or block cytokine signal transduction. Unfortunately, no structural data on the intracellular parts of cytokine receptors are available. The exact molecular mechanism underlying one of the first steps in signal transduction, namely the recruitment of signalling components to the cytoplasmic parts of cytokine receptors, remains elusive. Here we investigated possible mechanisms underlying the different potency of the STAT3-activating motifs of gp130 after IL-6 stimulation. Our data indicate that the extent of STAT3 activation by the different receptor motifs is not influenced by structural features such as contacts between the two gp130 chains. In addition, the proximity of the negatively regulating motif around tyrosine Y759 to the different STAT3-recruiting motifs does not seem to be responsible for their differential capacity to activate STAT3. However, the potency of a specific motif to activate STAT3 directly reflects the affinity for the binding of STAT3 to this motif.

The multi-site docking protein Gab1 is constitutively phosphorylated independent from its recruitment to the plasma membrane in Jak2-V617F-positive cells and mediates proliferation of human erythroleukaemia cells.

The constitutively active Janus kinase 2 mutant Jak2-V617F is responsible for cytokine-independent growth of hematopoietic cells and the development of myeloproliferative neoplasms, such as polycythaemia vera and essential thrombocythaemia. Cells expressing Jak2-V617F exhibit constitutive STAT, MAPK, and PI3K signalling, and constitutive association of the multi-site docking protein Gab1 to PIP3 at the plasma membrane. Here, we demonstrate the crucial role of Gab1 for the proliferation of Jak2-V617F-positive human erythroleukaemia (HEL) cells. In Jak2-V617F-expressing cells Gab1 is constitutively phosphorylated by Erk1/2 on serine residue 552, which regulates binding to PIP3. Additionally, Gab1 is constitutively phosphorylated on tyrosine residue 627. Tyrosine 627 is a SHP2 binding site and required for Gab1-dependent Erk1/2 activation. As previously shown, Jak2-V617F-dependent Erk1/2 and PI3K activation act synergistically on the proliferation of Jak2-V617F-positive cells. Here, we examined whether constitutive membrane association of Gab1 explains cytokine-independent Gab1 phosphorylation in Jak2-V617F-expressing cells. Although we could demonstrate Jak2-V617F-dependent constitutive serine 552 and tyrosine 627 phosphorylation of Gab1, interestingly, both phosphorylations do not require binding of Gab1 to PIP3 at the plasma membrane. Instead, we observed a constitutive interaction of Gab1 with the erythropoietin receptor in Jak2-V617F-expressing cells, which depends on Janus kinase activity. Thus, constitutive Gab1-dependent signalling in Jak2-V617F-expressing cells does not occur due to the constitutive association of Gab1 with PIP3 at the plasma membrane.